The invention relates to the technical field of the expression of the gdh and hex genes of Penicillium chrysogenum and of the act gene also of P. chrysogenum and of Acremonium chrysogenum. From analysis of the nucleotide sequence of said genes the existence of a promoter region which includes the translation initiation site, and which can be used to construct powerful expression and secretion vectors that are useful both for P. chrysogenum and for A. chrysogenum and related species, is deduced. In addition, these promoters can be used to block gene expression by means of antisense constructs. The expression of other genes in filamentous fungi can be directed under the control of the aforesaid promoters, with the production of antibiotics and/or proteins inherent therein being increased.
P. chrysogenum and A. chrysogenum are filamentous fungi which are of industrial interest because of their ability to produce penicillin and cephalosporin, respectively. During the last decade there has been considerable development of genetic manipulation techniques applicable in both microorganisms. The techniques for genetic manipulation of P. chrysogenum and A. chrysogenum include the transformation of protoplasts with vectors which use the phleomycin resistance gene (hereinafter called bleR gene) (Kolar, M. et al. (1988), Gene 62, 127-134) as a selection marker, as well as the expression of additional intact copies of genes of interest and the replacement of the promoter of the gene in question by another promoter which is able to improve its expression. The expression of homologous genes in fungi such as P. chrysoqenum or A. chrysogenum can be negatively regulated, whereas in the case of heterologous genes it is possible that their promoter may not be efficiently recognized by the said fungi. With the aim of avoiding these problems, genes were identified and cloned which are expressed constitutively and in which the said expression preferably does not show negative catabolic regulation, called hereinafter strong promoters. In general it is considered that the high-expression genes have signals in the promoter region which facilitate high transcription levels and which play a fundamental rxc3x4le in functions implicated in primary cellular metabolism. These genes include: the genes which code for NADP-dependent glutamate dehydrogenase (EC.1.4.1.4) (hereinafter called gdh gene), xcex2-N-acetylhexosaminidase (EC.3.2.1.52) (hereinafter called hex gene) and xcex3-actin (hereinafter called act gene).
There are earlier references to the gdh, hex and act genes from microorganisms other than those which are used in the present invention. The most relevant bibliography includes: (I) the nucleotide sequence of the gdh gene of the fungus Neurospora crassa (Kinnaird, J. H. and Fincham, J. R. S. (1983), Gene 26, 253-260) as well as the regulation of the expression of the gdhA gene of Aspergillus nidulans (Hawkins, A. R. et al. (1989), Mol. Gen. Genet. 418, 105-111), (II) the cloning and expression of the hex1 gene of Candida albicans (Cannon, R. D. et al. (1994), J. Bacteriol. 2640-2647) and (III) the characterization of the act gene of A. nidulans (Fidel, S. et al. (1988), Gene 70, 283-293). The expression of heterologous genes in P. chrysogenum using the promoters of the pcbC or penDE genes was described by Cantwell, C. A. et al. in 1992 (Proc. R. Soc. London Ser. B 248, 283-289). In addition, the expression of heterologous genes in A. chrysogenum using the promoters of the xcex2-isopropyl malate dehydrogenase gene (Japanese Patent Laid Open Publication No. 80295/1989) and glyceraldehyde 3-phosphate dehydrogenase gene (European Patent Application 0376226A1/1989) has also been described.
The inactivation of gene expression in industrial strains is sometimes necessary for the elimination of undesirable enzyme activities. Owing to the fact that the level of ploidy of many industrial strains makes it difficult in most cases to block expression by direct gene disruption, it is necessary to use systems for inactivation of expression which are independent of the level of ploidy. The development of antisense constructs expressed under the control of strong promoters makes interruption of gene expression possible. Constructs of this type are especially useful in industrial strains owing to the fact that their levels of ploidy (Kxc3xcnkel et al. (1992) Appl. Microbiol. Biotech. 36, 499-502) make it difficult to obtain complete gene inactivation. The use of antisense constructs for blocking enzyme activities has been described in yeasts (Atkins, D. et al. (1994), Biol. Chem. H-S 375, 721-729) and plants (Hamada, T. (1996), Transgenic Research 5, 115-121; John, M. E. (1996) Plant Mol. Biol. 30, 297-306). The hex promoter has the special feature of coding for an extracellular enzyme, which allows it to be used for the expression of extracellular proteins.
There are no citations in the prior art, however, which describe either the gene sequences of the filamentous fungi used in the present invention or those of the enzymes synthesized by the expression thereof. Nor is there any description in said Prior Art of the use of the strong promoters of the genes of the fungi described in the present invention for the expression, secretion or inactivation of gene expression.
The use of strong promoters to overexpress certain genes can lead to improvement in the production of penicillin or cephalosporin, and also to the synthesis of new antibiotics derived from the latter.
This invention describes a new process for obtaining strains of P. chrysogenum and A. chrysogenum with the ability to express homologous or heterologous genes under the control of strong promoters. The characterization and subsequent use of the promoters corresponding to the genes which code for NADP-dependent glutamate dehydrogenase (EC.1.4.1.4)xe2x80x94gdh genexe2x80x94of P. chrysogenum, xcex2-N-acetylhexosaminidase (EC.3.2.1.52)xe2x80x94hex genexe2x80x94of P. chrysogenum and xcex3-actinxe2x80x94act genexe2x80x94of P. chrysogenum and A. chrysogenum are described. The use of said promoters to overexpress genes related to the biosynthesis of penicillin and/or cephalosporin in the above-mentioned strains is one of the aims of the present invention. These promoters can also be used to block gene expression by means of antisense constructs.
The present invention is based on P. chrysogenum and A. chrysogenum as nucleic acid donors. Once the genomic DNA had been purified, DNA libraries of both microorganisms were constructed as described in Examples 1 and 4, and they were screened with: (I) synthetic oligonucleotides corresponding to the gdh gene of N. crassa in order to clone the homologous gene of P. chrysogenum, (II) combinations of oligonucleotides synthesized on the basis of the amino terminal sequence of the enzyme xcex2-N-acetylhexosaminidase in order to clone the hex gene of P. chrysogenum and (III) a fragment of the act gene of A. nidulans in order to clone the homologous genes of P. chrysogenum and A. chrysogenum. The clones purified by virtue of their ability to generate positive hybridization with the corresponding probe were subsequently analysed, the presence of the genes sought being determined.
The gdh gene of P. chrysogenum was identified in a 7.2 kb EcoRI fragment and in two BamHI fragments of 2.9 and 1.5 kb respectively. The restriction map of the DNA region which includes it is shown in FIG. 1. The 2,816 nucleotide sequence (SEQ ID NO:1) was then determined, which includes an open reading frame (ORF) with a very marked preferential codon usage pattern, the ATG translation initiation codon of which was found in position 922 and the TAA translation termination codon in position 2,522. The presence of 2 introns of 159 bp and 56 bp was also determined between positions 971-1130 and 1262-1318 respectively. Said ORF codes for a protein of 49,837 Da, with an isoelectric point of 6.18, the 461 amino acid sequence of which (SEQ ID NO:5) has 72.4% identity with the amino acid sequence of the NADP-dependent glutamate dehydrogenase enzyme of N. crassa. In the promoter region there are found pyrimidine-rich zones similar to those which appear in highly expressed genes, as well as two presumed TATA boxes (this box is found in certain promoters of fungi 30 to 50 bp upstream from the site of transcription initiation) (Davis, M. A. and Hynes, M. J. (1991), More Gene Manipulations in Fungi, Academic Press,. San Diego, Calif.) and a CCAAT box (which is found in about 30% of promoters of eukaryotic genes 50 to 200 bp upstream from the site of transcription initiation) (Bucher, P. (1990) J. Mol. Biol. 212: 563-578). This promoter was then used to express in P. chrysogenum and A. chrysogenum the E. coli gene which codes for xcex2-galactosidase (hereinafter called lacZ gene) and the bleR gene of S. hindustanus. The plasmids pSKGSu and pALfleo7 (FIG. 5) were constructed for this purpose, as described in Example 1. From-the results obtained it is deduced that the gdh promoter (hereinafter called Pgdh) is able to control the expression of the heterologous lacZ and bleR genes both in P. chrysogenum and A. chrysogenum and also in E. coli. 
The development of antisense constructs expressed under the control of strong promoters makes the interruption of gene expression possible. The plasmid pALP888 (FIG. 5) was constructed for this purpose, as described in Section 1.3 of Example 1. The results obtained confirm the possibility of totally or partially blocking undesirable enzyme activities in P. chrysogenum by the use of antisense constructs using Pgdh.
The hex gene of P. chrysogenum was identified in a 3.2 kb SacI fragment and in a 2.1 kb SalI fragment. The restriction map of the DNA region which includes the hex gene is shown in FIG. 2. The 5,240 nucleotide sequence (SEQ ID NO:2) was then determined, confirming the existence of two ORFs with a very marked preferential codon usage pattern, one of which matched the hex gene. The ATG translation initiation codon of the hex gene was found in position 1,324 and the TGA termination codon in position 3,112. Said ORF has no introns and codes for a protein of 66,545 Da, with an isoelectric point of 5.34, the 596 amino acid sequence of which (SEQ ID NO:6) has 49.0% identity with the amino acid sequence of the xcex2-N-acetylhexosaminidase enzyme of Candida albicans. In addition, the deduced amino acid sequence includes the polypeptides determined chemically from the purified enzyme in positions 19-40 and 99-120. In the promoter region there are found two pyrimidine-rich zones, a presumed TATA box and the CAAT box. This promoter was then used to express the bleR gene of S. hindustanus in P. chrysogenum. The plasmid pALP480 (FIG. 6) was constructed for this purpose, as described in Example 2. From the results obtained it is deduced that the hex promoter (hereinafter called Phex) is able to control the expression of the heterologous bleR gene in P. chrysogenum. In addition, the fact that the enzyme xcex2-N-acetylhexosaminidase is a protein abundantly secreted by P. chrysogenum to the culture medium makes it possible to use the hex gene for the expression and secretion of homologous or heterologous proteins in P. chrysogenum or related filamentous fungi. The genes to be expressed can be fused in a reading frame with the promoter region, including the secretion signal sequence of the hex gene, or else they can be fused in a reading frame with the complete hex gene.
The act gene of P. chrysogenum (hereinafter called actPc) was identified in a 5.2 kb BamHI fragment, a 4.9 kb EcoRI fragment and a 5.9 kb HindIII fragment. The restriction map of the DNA region which includes the actPc gene is shown in FIG. 3. Once the 2,994 nucleotide sequence (SEQ ID NO:3) had been determined, the existence of an ORF with a very marked preferential codon usage pattern was confirmed. The ATG translation initiation codon was found in position 494 and the TAA termination codon in position 2,250. Said ORF has 5 introns and codes for a protein of 41,760 Da, with an isoelectric point of 5.51, the 375 amino acid sequence of which (SEQ ID NO:7) has 98.1% identity with the amino acid sequence of the xcex3-actin protein of A. nidulans. In the promoter region there are found two pyrimidine-rich zones, a presumed TATA box and four CAAT boxes. This promoter was then used to express the bleR gene of S. hindustanus in P. chrysogenum. The plasmid pALPfleo1 (FIG. 6) was constructed for this purpose, as described in Example 3. From the results obtained it is deduced that the act promoter of P. chrysogenum (hereinafter called PactPc) is able to control the expression of the heterologous bleR gene in P. chrysogenum. 
The act gene of A. chrysogenum (hereinafter called actAc) was identified in SalI fragments of 2.4 and 1.1 kb, a 3.9 kb SmaI fragment and an 8.7 kb HindIII fragment. The restriction map of the DNA region which includes the actAc gene is shown in FIG. 4. The 3,240 nucleotide sequence determined (SEQ ID NO:4) confirmed the existence of an ORF with a very marked preferential codon usage pattern. The ATG translation initiation codon was found in position 787 and the TAA termination codon in position 2,478. Said ORF has 5 introns and codes for a protein of 41,612 Da, with an isoelectric point of 5.51, the 375 amino acid sequence of which (SEQ ID NO:8) has 98.4% and 98.1% identity with the amino acid sequences corresponding to the xcex3-actin proteins of A. nidulans and P. chrysogenum, respectively. In the promoter region there are found pyrimidine-rich zones and a CAAT box, the existence of a TATA box not being observed. This promoter was then used to express the bleR gene of S. hindustanus in A. chrysogenum. The plasmid pALCfleo1 (FIG. 6) was constructed for this purpose, as described in Example 4. From the results obtained it is deduced that the act promoter of A. chrysogenum (hereinafter called PactAc) is able to control the expression of the heterologous bleR gene in A. chrysogenum. 
In all cases, the expression of the heterologous gene in P. chrysogenum or A. chrysogenum under the control of the fungal promoter was achieved by fusing the said gene in the correct reading frame. Although the lacZ and bleR genes were expressed by way of example, it would be possible in the same way to express genes which code for enzymes involved in the biosynthesis of penicillin: pcbAB (xcex1-aminoadipyl-cysteinyl-valine synthetase), pcbC (isopenicillin N synthase), penDE (acyl-CoA:6-APA acyltransferase), pcl (phenylacetyl-CoA ligase), etc.; or of cephalosporin: pcbAB (xcex1-aminoadipyl-cysteinyl-valine synthetase), pcbC (isopenicillin N synthase), cefD (isopenicillin N isomerase), cefEF (deacetoxycephalosporin C synthase/hydroxylase), cefG (deacetylcephalosporin C acetyltransferase), etc. The gene to be expressed may have been obtained by different methods: isolated from chromosome DNA, cDNA synthesized from mRNA, synthesized chemically, etc. The fundamental processes for correct promoter-gene fusion are described in Sambrook, J. et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA, and Ausubel et al. (1987), Current Protocols in Molecular Biology, John Wiley and Sons, New York, USA.
P. chrysogenum and A. chrysogenum were used as host strains, but any related strain or mutant strain derived from them can be used. The process employed for the production of protoplasts and transformation of P. chrysogenum was based on that described by Cantoral et al. in 1987 (Biotechnology 5: 494-497) and Dxc3xadez et al. in 1987 (Curr. Genet. 12: 277-282) and is described in Example 1. The production of protoplasts and transformation of A. chrysogenum are described in Example 4. In both cases use was made of the antibiotic phleomycin as selection marker and the plasmids pALfleo7, pALP480, pALPfleo1 or pALCfleo1, which are carriers of the bleR gene expressed under the control of Pgdh, Phex, PactPc and PactAc, respectively. It would be possible, however, to use any marker which can selectively separate the transformant strains from the others, which are not.
The transformant may be grown in culture media containing carbon and nitrogen sources which can be assimilated. Examples of carbon sources are glucose, sucrose, lactose, starch, glycerine, organic acids, alcohols, fatty acids, etc., used alone or in combination. Examples of nitrogen sources would be peptone, malt extract, yeast extract, corn steep liquor, gluten, urea, ammonium salts, nitrates, NZ-amine, ammonium sulphate, etc., used alone or in combination. Inorganic salts which can be used as components of the culture medium include phosphates (for example potassium phosphate), sulphates (for example sodium sulphate), chlorides (for example magnesium chloride), etc., and iron, magnesium, calcium, manganese, cobalt, etc., can be used as ions. The cultural conditions such as incubation temperature, pH of the culture medium, aeration, incubation time, etc., must be selected and adjusted in accordance with the strain used. In general terms, however, fermentation is carried out for a period of 4 to 14 days under aerobic conditions at a temperature between 20xc2x0 C. and 30xc2x0 C. and a pH between 5 and 9.
In summary, the present invention includes: (I) DNA fragments which contain the promoters of the gdh, hex and act genes of P. chrysogenum and of the act gene of A. chrysogenum, (II) plasmids which incorporate the aforesaid promoters together with their translation initiation site, (III) plasmids in which a homologous or heterologous structural gene or an antisense DNA fragment is situated, under the control of the said promoters, (IV) P. chrysogenum or A. chrysogenum strains transformed with said plasmids, (VI) transformant strains able to express the structural gene or the antisense DNA situated in the plasmid under the control of the promoter and (VII) transformant strains able to secrete homologous or heterologous extracellular proteins under the control of the Phex.